1. Field of the Invention
The present invention relates generally to methods and apparatus for focusing and mobilizing solutes of an aqueous solute mixture. More particularly, the present invention involves the mobilization of focused protein zones past a detector associated with a capillary isoelectric focusing apparatus without relying upon the application of pressure, vacuum, or buffer changes.
2. Description of Relevant Art
Recently capillary isoelectric focusing ("IEF") techniques have emerged as a powerful tool for rapidly separating, analyzing and quantitating small quantities of proteins. These techniques are based upon traditional isoelectric focusing principles and are carried out in free solution of amphoteric buffers within a length of capillary tubing. Capillary isoelectric focusing is a form of capillary electrophoresis which separates or fractionates proteins according to their isoelectric point ("pI"). Typically, protein focusing processes are performed in a medium containing a mixture of ampholytes which form a pH gradient extending along the length of the capillary in the presence of an electric field. The IEF process involves applying an electric field through the ampholytes along the interior of the capillary. Under the influence of the electric field, the solutes at one end of the capillary migrate along the capillary length until each solute reaches a location at which the pI of the solute corresponds to the local pH (i.e. a location of zero net charge).
After the migration is complete and all the solutes are stabilized at their respective focused zones, the solute zones are mobilized past a detector to determine the relative migration distance or time of each solute. Then, from plots of elution time versus pI of known solutes focused according to the same conditions, the pI corresponding to each unknown protein is determined.
Since IEF is a process in which the solutes seek their "focus" position in a separation medium, it is important that the medium remains stationary during migration of the solutes to their respective positions in the medium. A phenomenon which frustrates this objective is electroosmosis flow ("EOF") which inherently occurs in electrophoresis being carried out in silica containing capillary tubes. This phenomenon arises from a potential, termed the zeta potential, which develops between the inner surface of the silica tubing and a diffuse layer in the buffer adjacent the inner surface. More particularly, the silica capillary tubing surface tends to ionize to form an anion rich surface, and the diffuse layer carries cationic counter-ions to the anions on the ionized surface. In the presence of an electric field along the buffer, the charged buffer migrates to the anode or cathode. The exact direction of buffer flow and the rate of solvent flow within the capillary depend on the polarity and magnitude of this potential.
While it is desirable to maintain the medium stationary during IEF, it is also desired that the medium be mobilized after IEF for analysis of the separated solutes carried therein. Accurate and precise detection results depend upon mobilizing or moving the focused zones carried in the medium past a suitable detector while preserving the spatial relationship of the protein separation zones as focused within the capillary.
A number of different methods for mobilizing focused solutes in order to detect their stabilized locations within the buffer gradient have been suggested. For example, one method involves pumping a solution through the capillary to move the zones by hydrodynamic flow. A related method utilizes a vacuum technique to pull the zones past a suitable detector. A major problem associated with each of these methods is that hydrodynamic flow is characterized by a parabolic flow profile which causes zone broadening or distortion, thereby affecting accuracy and precision of the analytical process.
Another method for mobilizing focused proteins is disclosed in U.S. Pat. Nos. 4,911,808 and 4,725,343 and involves producing a pH shift in the separation medium within the column subsequent to focusing the mixture components. This effectively restores a charge to the ampholytic buffers and causes them to move under the influence of the internally applied voltage. Unfortunately, at zones where the pH gradient is characterized by very high or very low pI's, band broadening and non-linear effects occur during the mobilization. This in turn results in analytical error for proteins detected within these extreme zones. Additionally, many focusing procedures may be limited in pH by the instability of the separated molecules or by the alteration of charges on the molecules which alter their electrophoretic mobilities.
To this date, no effective means of EOF control as well as solute mobilization in an IEF process have been devised.
Accordingly, it is an objective of the present invention to provide an improved IEF process which incorporates means for controlling EOF during IEF.
It is another objective of the present invention to provide an effective means for mobilizing isoelectrically focused solutes for detection after IEF while avoiding hydrodynamic flow and associated band broadening.
It is further an objective of the present invention to couple EOF control and solute mobilization by using the same means.